Recently, various saccharide-related enzymes which synthesizes certain oligosaccharides were screened for on the basis of microorganisms, and the advancement in a technology for utilizing such a microorganism-derived enzyme and in a technology for purifying a resultant synthetic oligosaccharide extensively enables a large scale production of the oligosaccharide at a low cost. Accordingly, an oligosaccharide became utilizable even in a field familiar to us such as a food industry. Those known typically are a coupling sugar, fractooligosaccharide, β-galactooligosaccharide, soybean oligosaccharide, isomaltooligosaccharide, palatinose, lactosucrose oligosaccharide and the like.
Any of these oligosaccharide was reported to have a bifidus microorganism growth activity or a cariostatic or anti-cariogenic property as well as a mineral absorption-promoting effect, and is utilized as a specified health-care food or a starting material thereof.
It is also reported recently that an oligosacchride containing α-galactosyl such as melibiose, manninotriose, raffinose and stachyose has a potent proliferating effect on a bifidus microorganism, has a favorable property as a food, and have a carcinostatic effect or natural killer cell-activating effect (Shigeki Motoi et al, Japanese Cancer Association (meeting report), 40, 132 (1981)), and such an oligosaccharide is considered to be extremely useful and attractive as a beverage, food product, pharmaceutical and a starting material thereof.
A known source of raffinose is a beet or soybean oligosaccharide, while that of stachyose is a soybean oligosaccharide. However, a soybean oligosaccharide is not found in abundance, and can not be supplied in a large amount. Besides, it is costly. Raffinose contained in a beet is also disadvantageous since it is available only during a period of October to March, its annual supply is only several 100 tons and its current price is so high. Melibiose and manninotriose are present in small amounts in a soybean oligosaccharide, and have the structures formed by deleting a fructose moiety from raffinose and stachyose, respectively. While they are synthesized artificially by means of the decomposition of raffinose and stachyose, they are very expensive since the starting raffinose and stachyose are also expensive.
On the other hand, an enzyme reaction employing an α-galactosidase may also be employed for synthesizing an oligosacchride containing α-galactosyl. Such an enzyme reaction is classified into a saccharide transfer reaction and a dehydrocondensation reaction.
The saccharide transfer reaction is not suitable as an industrial method since it requires a synthesis substrate such as p-nitrophenyl α-galactoside as an α-galactosyl group donor (JP-A-10-201472) and also requires as a substrate an expensive compound containing α-galactosyl such as raffinose.
When utilizing the dehydrocondensation reaction, it is possible to conduct a reaction at a high substrate concentration, and a free galactose employed as a substrate may be one obtained by a hydrolysis of a less expensive saccharide such as lactose, whereby accomplishing an economically advantageous reaction.
An enzyme which can be employed in this dehydrocondensation reaction is proposed to be an α-galactosidase produced by a microorganism such as Pycnoporus cinnabarinus, Streptococcus bovis, Diplococcus pneumoniae, Mortierella vinacea, Pseudomonas fluorescens strain H-601 (deposition No: FERM P-11027) and Candida guilliermondii strain H-404 (deposition No: FERM P-11026) as well as a plant such as Vicia sativa and Green coffee bean (Japanese Patent No. 3028258).
However, these enzymes are not applied to an industrial scale production since they exhibit low reaction rates and low reaction yields at high substrate concentrations. Thus, the α-galactosidase produced by Candida guilliermondii strain H-404 which has been believed to exhibit the highest reaction rate and the highest reaction yield requires a period, for example, as long as about 90 hours until its reaction is plateaued when being treated with 60% (w/v) galactose under the conditions of 35 UM/g-galactose and 50° C., but only gives an yield of oligosacchrides containing α-galactosyl as low as about 25% (Applied Science of Saccharide, 44, 69-75, 1997).
As described above, an oligosacchride containing α-galactosyl having useful characteristics as a food material is not supplied in a large amount or at a low cost and desired to be produced on an industrial scale.
The invention is intended to solve the problem that a conventional method poses a low reaction rate and a low reaction yield in a dehydrocondensation reaction at a high substrate concentration and is less practical, and its objective is to provide a method for producing an oligosacchride containing α-galactosyl far more efficiently than any conventional method by using a certain α-galactosidase.
On the other hand, a Candida microorganism is one of the yeasts which are persistent in the intestine of a human, and is an opportunistic pathogen inducing a serious systemic infections when the resistance is reduced due to an infectious disease or other diseases. An intestinal Candida microorganism exerts its effect on an immune system via the cell itself or a Candida toxin produced by the microorganism, and was reported to serve as an atopic dermatitis exacerbation factor (Michio Matsuda et al., allergy clinics, 56, 768-772 (1991)) or to be involved in the allergic diseases other than the atopic dermatitis, such as bronchial asthma and rhinitis. Otherwise, it induces, when proliferating abnormally in an intestine, a health problem such as a chronic fatigue called yeast connection, headache, uneasiness, unsettledness, depression and the like.
As a method for treating such a Candida-induced mycosis or atopic dermatitis, an antifungal therapy is considered to be effective, and it was reported recently that a trisaccharide raffinose occurring in a beet plant has not only in vitro but also in vivo inhibitory and eradicative effects on a Candida microorganism (JP-A-11-240837) and actually exerted an effect on an atopic dermatitis (Taizo Nagura et al, Food Industries, 2.28, 29, 1999). An oral administration of raffinose does not cause side effects observed with an antifungal agent such as a transient exacerbation of rash or digestive organ signs, and is regarded as a safer therapy against an atopic dermatitis.
Nevertheless, a further potent and further safer therapy has been demanded since the anti-candida effect of raffinose is lower than that of an antifungal agent. Also since a Candida microorganism has various adverse effects on a human health as described above, a food product having a more potent anti-candida effect has been desired from a viewpoint of a diet-based health control trend in response to the demand of reducing the medical expense. Another problem associated with raffinose is its limited supply and expensiveness as described above.
On the other hand, the physiological functions of an oligosacchride containing α-galactosyl other than raffinose were also reported intensively, including a neutralizing effect of α-1,3-galactobiose (α-1,3 Gal2) on a toxin produced by Clostridium difficile which is a causative bacteria of a colitis (L. D. Heerze et al., J.Infect.Dis, 169, 1291-1296 (1994)) and an inhibitory and therapeutic effect of α-1,4 galactobiose (α-1,4 Gal2) on a pathogenic E.coli 0157 (C. A. Lingwood, Adv. Lipid Res., 25, 189-211 (1993)).
Nevertheless, no reports suggested any anti-candida effect of an oligosacchride containing α-galactosyl other than raffinose.
Also since α-galactosyl group is present only in the form of an α-galactobiose (α-Gal2) in a living body and the α-1,6-galactosyl glucose structure possessed by raffinose does not exist while the mechanisms of the anti-candida effect of raffinose are considered to be based on the intestinal flora-improving effect, the inhibitory effect on the adhesion or settlement of a Candida microorganism on a digestive tract and the immune activation effect, we assumed that α-Gal2 plays an important role in the in vivo effect of raffinose and focused on an oligosacchride containing α-galactosyl other than raffinose, whereby accomplishing the invention.
Accordingly, an objective of the invention is to provide an anti-candida composition which is derived from a food, highly safe, excellent in anti-candida effect, and can be supplied limitlessly.